Methods of identifying organism based on its genotype

ABSTRACT

A method for identifying a microorganism, wherein the method comprises the steps of: 1) preparing one or more kind(s) of double-stranded DNA fragments by the random PCR using at least a part of a genome of an organism of interest, 2) applying the double-stranded DNA fragments which were prepared in step 1 to temperature gradient gel electrophoresis (TGGE) or denaturant gradient gel electrophoresis (DGGE), 3) extracting identification dots of each DNA fragment from the electrophoresis pattern which was obtained in step 2, 4) determining PaSS and/or genome semi-distance from the identification dots which were obtained in step 3, and 5) analyzing the PaSS and/or genome semi-distance which were/was obtained in step 4, wherein pseudo-absolute position of identification dots is determined by the locational relation to standard DNA in the presence of standard DNA as the starting dots in TGGE or DGGE.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a method for identifying a species orhomology of an organism such as microorganism by its genotype.

2. Description of the Related Art

Organisms which include microorganisms have been identified so farbasically using their phenotypes. However, identification by phenotypeswas not suited for distinguishing organisms more accurately.Particularly, there was practical limitation upon identifying/specifyingmicroorganisms comprising a large number of species by phenotypes. Ourdaily life is surrounded by microorganisms. Microorganisms such asEscherichia coli 0157, Mycobacterium tuberculosis, methicillin-resistantStaphylococcus aureus (MRSA), and Vibrio cholera cause many diseases,and a technique for accurately identifying microorganisms is needed forestablishing effective treatments and specifying infection pathways. Itis also recognized that soil bacteria are involved in the productivityand quality of an agricultural product, and that the human health isgreatly influenced by the enteric bacterial flora. Accurate studies havenot carried out, however, concerning the relation between theproductivity or quality of an agricultural product and the kind,quantity, and combination of soil bacteria, and the relation between thehuman health and the enteric bacterial flora. This is due to the factthat it is impossible to accurately identify/distinguishmicroorganism(s) by the conventional identification by its/theirphenotype(s).

In the above-mentioned situation, identification of microorganisms bygenotypes is proposed instead of that of phenotypes.

It is quite possible to identify/distinguish microorganism(s) bycomparing the (whole) genome of each microorganism using the recentsequencing technique level. It needs, however, a significant amount ofwork and time, and is not a simple method. Therefore,identifying/distinguishing species of microorganisms by comparing the(whole) genome is practically not a widely applicable method. Although amethod for comparing a part of a genome is known as a more simplemethod, enough information is not obtained foridentifying/distinguishing species by comparing the 16S rRNA sequences.

SUMMARY OF THE INVENTION

It is therefore the object of the present invention to provide a methodfor identifying the species, homology, and so on of organisms such asmicroorganisms, which is simple to some extent and practically workable,and is based on genotypes.

The present invention is directed to a method for identifying anorganism comprising the steps of:

-   1) preparing one kind or more of double-stranded DNA fragments by    the random PCR using, as a template, at least a part of a genome of    an organism which is to be identified,-   2) subjecting the double-stranded DNA fragments prepared in step 1    to temperature gradient gel electrophoresis (TGGE) or denaturant    gradient gel electrophoresis (DGGE),-   3) extracting identification dots of each DNA fragment from an    electrophoretic pattern which was obtained in step 2,-   4) determining PaSS and/or genome distance(s) from the    identification dots which were obtained in step 4, and-   5) analyzing the PaSS and/or genome semi-distance(s) which was/were    obtained in step 4,-   wherein in the electrophoresis by TGGE or DGGE, a standard DNA is    co-existed as a standard point for the identification dots and the    pseudo-absolute location of the identification dots is determined    from the locational relation to the standard DNA.

BRIEF DESCRIPTION OF THE DRAWINGS

The above and other objects, aspects, features and advantages of thepresent invention will become more apparent from the following detaileddescription when taken in conjunction with the accompanying drawings, inwhich:

FIG. 1 illustrates examples of genome profiling images (an electrophoresis pattern which is obtained by TGGE of standard DNAs which are shown bySeq. ID Nos. 1 and 2);

FIG. 2 illustrates the first typical band curve;

FIG. 3 illustrates the second typical band curve;

FIG. 4 illustrates eight featuring points P_(j) (j=1-8) which wereextracted from three band curves;

FIG. 5 illustrates ‘n’ featuring points P_(1i) (i=1−n) with respect to aspecimen microorganism which corresponds to ‘n’ featuring points P_(0i)(i−1−n) with respect to a reference genome; and

FIG. 6 a illustrates an electrophoresis pattern in case cy3-pfM12 (5′cy3-AGA ACG CGC CTG 3′) (SEQ ID NO: 4) which was obtained in Example 2was used as a primer, and FIG. 6 b illustrates an electrophoresispattern in case FITC/UCS (5′ FTTC-CA GGA AAC AGC TAT GAC 3′) (SEQ ID NO:38)was used as a primer.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

(1) Preparation of Double-stranded DNA Fragments from Genome by RandomPCR

The random PCR is a method which was developed by the inventors.

The normal polymerase chain reaction (PCR) method is a method foramplifying a specific DNA region by repeating DNA synthesis using twokinds of primer which sandwich a specific DNA region and DNA polymerase.Specifically, 1) a double-stranded DNA is denatured by heat treatment at90° C. or so to give single-stranded DNAs (denaturing), 2) a primerattaches to the obtained single-stranded DNA by annealing at 50° C. orso, and 3) a DNA chain is synthesized by DNA polymerase using the primeras the starting point using the single-stranded DNA as a template usingmonomer nucleotides as raw materials at 65° C. or so (elongation ofstrand). Only a double-stranded DNA fragment is amplified whichcorresponds to a sequence in a specific DNA region which was sandwichedbetween parts which have sequences which are complementary to the twokinds of primer by repeating a cycle comprising the denaturing, theannealing, and the elongation.

The above-mentioned PCR method is a method for amplifying only a DNAfragment in a specific DNA region. Therefore, the condition for theabove-mentioned annealing is designed so that the primer can bind to aspecific part of a single-stranded DNA which has a sequence which iscomplementary to the sequence of the primer (e.g., a pair of G and C ora pair of A and T). Namely, the condition for the annealing is designedso that a primer and a single-stranded DNA can form a double strandwithout causing a mismatch between the primers and the single-strandedDNA as a template. DNA strand can be amplified even by carrying out theabove-mentioned annealing at about 30° C. and carrying out theelongation of strand at about 50° C. However, the temperature for theannealing is low, so that mismatch(s) (i.e., not complementary; e.g., apair comprising G and A) can be contained in a part of the base pairsbetween a primer and a single-stranded DNA as a template. Normally, onlya DNA fragment which has a absolutely complementary sequence can be aprimer and allows amplifying the strand. On the contrary, if theannealing and the elongation are carried out at a relatively lowtemperature as described above, even a DNA fragment which does not havea completely complementary sequence can be a primer and allowsamplifying the stand.

Therefore, if a cycle comprising denaturing, annealing, and elongation(wherein annealing and elongation are carried out at a lower temperaturethan the normal PCR) is repeated using DNA such as genome DNA as atemplate using a DNA fragment which has a specific sequence (e.g., afragment which is 12mer or so, and is selected independent of a DNAsequence of a template) as a primer, even a DNA fragment which does nothave a completely complementary sequence can be a primer, and the strandis amplified, so that one kind or more of double-stranded DNA fragmentwhich can contain mismatch (es) in a primer part is/are formed. Thepurpose of this method is not to amplify a specific region on atemplate, and a strand is amplified if it can be a starting point forthe elongation even if mismatches are contained to some extent, so thattwo kinds or more of double-stranded DNA fragment can be formed. Inaddition, it is found that if the same DNA which is used as the templateand the same DNA fragment which is used as a primer and the same cycleconditions for denaturing and annealing are used, DNA strand(s) whichhas/have the same sequence(s) is/are always amplified. This method iscalled ‘Random PCR’ because the purpose of the method is not to amplifya specific region on a template.

By the random PCR, various DNA strands can be obtained in amplificationby using a primer DNA fragment different in a sequence even if the samegenome DNA is used as a template, or by changing the condition for thecycle of denaturing, annealing, and elongation. Especially obtained DNAstrand(s) is/are largely changed by both length and sequence of theprimer, so that it is preferable to choose an appropriate primer.Versatile sequence information of a genome can be taken out by changinga nucleotide sequence of a primer taking advantage of the character.

A method according to the present invention allows amplifying andpreparing one or more kind(s) of double-stranded DNA fragment by therandom PCR using as a template at least a part of a genome of amicroorganism whose species and other properties are to be identified.At least a part of a genome which is used as a template in the randomPCR can be prepared from a microorganism according to generally usedmethods. At least a part of a genome which is used in the random PCR asa template can be prepared, for example, by the alkali method (H. Wang,M. Gin, A. J. Cutler, Nucl. Acids Res., 21, 4153 (1993)).

The random PCR in which an obtained genome is used as a template can becarried out as follows:

Apart from the normal PCR method, an appropriate sequence, length andkind of a primer can be appropriately selected for the random PCR.Experimentally and analytically the most simple system, however, is thecase where a relatively short single primer is used. Even in the case asingle primer is used, and a genome which is derived from the samemicroorganism is amplified, a kind and quantity of a double-stranded DNAfragment which can be amplified depend on the sequence and length of theprimer. In addition even in the case a single primer and a primer whichhas the same sequence and length are used, a kind and quantity of adouble-stranded DNA fragment which can be amplified depend on themicroorganism from which the genome is derived. Considering these facts,sequence, length, and kind of a primer are appropriately selected. Toolong primer can have the hair-pin structure, can prevent annealing, andtends to lower a yield of a PCR product. Too short primer does notadequately stabilize a bonding containing mismatch (es), and tends tolower a yield of a PCR product. A length (base length) of a primer issuitably, for example, 8-20, preferably 10-16, more preferably 10-12,and most preferably 12. An oligonucleotide which has a length of 12bases can be used as a primer. Such oligonucleotide primers include pfM4(dCAGTCAGGACGT), (SEQ ID NO: 3), pfM12 (dAGAACGCGCCTG) (SEQ ID NO: 4),pfM14 (dCGTCGCTATTAA) (SEQ ID NO: 5), pfM19 (dCAGGGCGCGTAC) (SEQ ID NO:6), (dA)₁₂ (dAAAAAAAAAAAA) (SEQ ID NO: 7), (dA₃T₃)₂ (dAAATTTAAATTT) (SEQID NO: 8), (dAATT)₃ (dAATTAATTAATT) (SEQ ID NO: 9), (dACG)₄(dACGACGACGACG) (SEQ ID NO: 10), (dAT)₆ (dATATATATATAT) (SEQ ID NO: 11),(dC)₁₂ (dCCCCCCCCCCCC) (SEQ ID NO: 12), (dCCGG)₃ (dCCGGCCGGCCGG) (SEQ IDNO: 13), (dG)₁₂ (dGGGGGGGGGGGG) (SEQ ID NO: 15), (dGA)₆ (dGAGAGAGAGAGA)(SEQ ID NO: 16), (dGGCC)₃ (dGGCCGGCCGGCC) (SEQ ID NO: 17), (dCT)6(dCTCTCTCTCTCT) (SEQ ID NO: 14), (dT)₁₂ (dTTTTTTTTTTTT) (SEQ ID NO: 18),(dT₃G₃)₂ (dTTTGGGTTTGGG) (SEQ ID NO: 19), (dTGC)₄ (dTGCTGCTGCTGC) (SEQID NO: 20), (TA)₄C₂AC (dTATATATACCAC) (SEQ ID NO: 21), Cohesive1(dGGGCGGCGACCT) (SEQ ID NO: 22), Cohesive2 (dAGGTCGCCGCCC) (SEQ ID NO:23), 4sand(dGGGGTCGAGGGG) (SEQ ID NO: 24), GCTA₉ (dGCTAAAAAAAAA), (SEQID NO: 25), notG (dCAATTCTACAAC) (SEQ ID NO: 26), notT (dACGAGCGAGCGC)(SEQ ID NO: 27), Promote1 (dTATAATTATAAT) (SEQ ID NO: 28), Promote2(dATTATAATTATA) (SEQ ID NO: 29), SD1 (dGATCACCTCCTTA) (SEQ ID NO: 30),SD2 (dTAAGGAGGTGATC) (SEQ ID NO: 31), Telomere1 (dCCCACCCACCCA) (SEQ IDNO: 32), Telomere2 (dTGGGTGGGTGGG) (SEQ ID NO: 33), FITC17-H-3′ (5′GAGGAAACAGCTATGAGATCT TCTC 3′), (SEQ ID NO: 34), FITC17-H-5′ (5′ CAG GAAACA GCT ATG ACG TTC TCA C 3′), (SEQ ID NO: 35), LH-7-3′ (5′ GGC GAT ATCCCT GAA A 3′), (SEQ ID NO: 36), LH-7-5′ (5′ TAT TAT TTC CGC AAA G 3′)(SEQ ID NO: 37), M13 Reverse (5′ CAG GAA ACA GCT ATG AC 3′) (SEQ ID NO:38), cy3-pfM12 (5′ cy3-AGA ACG CGC CTG 3′) (SEQ ID NO: 39) (with cy3fluorescence), FITC/UCS (5′ FITC-CA GGA AAC AGC TAT GAC 3′) (SEQ ID NO:40) (with FITC fluorescence), MA1-FITC (5′ FITC-TGC TAC GTC TCT TCC GATGCT GTC TTT CGC T 3′) (SEQ ID NO: 41) (with FITC fluorescence), cy3-MA1(5′ cy3-TGC TAC GTC TCT TCC GAT GCT GTC TTT CGC T 3′) (SEQ ID NO: 42)(with cy3 fluorescence), HEX-pfM11 (5′ HEX-GAA CCT CCC GAC 3′) (SEQ IDNO: 43) (with Hex fluorescence), TAM-TGC4 (5′ TAM-TGC TGC TGC TGC 3′)(SEQ ID NO: 44) (with Tamara fluorescence), and the like.

In the random PCR, as described above, PCR is operated at a remarkablylower temperature than the normal PCR while other operational conditionsfor the random PCR are substantially the same as the normal PCR.Reaction mixture and reaction condition for the random PCR are asfollows: 100 μl of reaction mixture contains 200 μM dNTP (N=G, A, T, C),0.5 μM primer, 10 mM Tris-HCl (pH 9.0), 50 mM KCl, 2.5 mM MgCl₂, 0.1%Triton X-100, 0.02 unit/μl Taq DNA polymerase, and an appropriate amount(e.g., 3 μl of a DNA solution which was prepared in a way which isdescribed elsewhere is added and the total volume of the reactionmixture was adjusted to 100 μl) of template DNA. PCR comprises 20-30cycles of the following steps: treatment at 94° C. for 1 min,denaturation at 94° C. for 30 min, annealing at 28° C. for 2 min, andelongation at 47° C. for 2 min, followed by treatment at 47° C. for 2min. When pfM14 which has a low G+C content is used as a primer,annealing can be carried out at 23° C., elongation can be carried out at42° C. for 10 min, and the final chase treatment can be carried out at42° C. for 10 min. For the experiment which was described above, it ispreferable to carry out preparation and mixing of the reaction mixtureon a clean bench, and to irradiate UV light (312 nm for 10 min) to asolution (which contains all except template DNA) immediately beforeadding template DNA to prevent formation of PCR products which arederived from impurities.

For the above-mentioned random PCR, DNA labeled with a fluorescentmarker can be amplified by using a raw material with a fluorescentmarker. In this case, using a fluorescence labeled DNA as a standardDNA, identification dots (as described below) can be extracted by meansof the image processing using fluorescence markers carried by the DNAs.A primer or nucleotide can be the raw material labeled with afluorescent marker. A primer or nucleotide labeled with a fluorescentmarker can be purchased or easily synthesized according to well knownmethods.

(2) Electrophoresis of Double-stranded DNA Fragment by TGGE or DGGE

According to the present invention, the double-stranded DNA fragmentsobtained in (1) are subjected to temperature gradient gelelectrophoresis (TGGE) or denaturant gradient gel electrophoresis(DGGE).

Both TGGE and DGGE are well known methods. TGGE is described in detail,for example, in R. Riesner, et al., Electrophoresis 10, 377-389 (1989).DGGE is described in detail, for example, in E. S. Abrams, V. P.Stanton, Jr., Methods Enzymol., 212, 71 (1992). Both methods can becarried out easily using commercial apparatus. Both TGGE and DGGE aretwo-dimensional electrophoresis using a slab-type gel providing atemperature gradient or denaturant concentration gradient along andvertically to the electrophoresis direction of the double-stranded DNA.TGGE and/or DGGE allows understanding the behavior of melting(dissociation of two strands) which is characteristic to the nucleotidesequence of the double-stranded DNA. Namely, the strength (heatresistance or denaturant resistance) of bond of base pairs in a regionwhich exists in the double-stranded DNA to be subjected toelectrophoresis can be visualized by TGGE or DGGE.

For TGGE, a temperature gradient from 30° C. to 70° C. is normallyapplied. For DGGE, a denaturant such as urea and formamide can be usedwith a concentration gradient, for example, from 0 to 15M.

For a method according to the present invention, the pseudo-absoluteposition of an identification dot is determined from the locationalrelation to standard DNA which is added as a standard point for theidentification dot upon electrophoresis by TGGE or DGGE. With thedouble-stranded DNA which was amplified by the random PCR, the standardDNA is co-existed as the standard dot for the identification dot, andthen the mixture is subjected to electrophoresis, whereby the standardDNA also give an electrophoresis pattern corresponding to its sequence.The pseudo-absolute position of the identification dot of eachdouble-stranded DNA can be determined from the locational relation tothe electrophoresis pattern of the standard DNA. The standard DNA can bea kind of an inner standard specimen. Using such standard DNA allowsanalyzing data, which are obtained by the electrophoresis and havedeviation to some extent depending on the condition, based on the samestandard. Namely, using standard DNA allows correcting data which areobtained from electrophoresis pattern and normalizing them.

In electrophoresis by TGGE or DGGE, at least a part of double-strandedDNA has the melting-starting dot, the slowest dot (a dot where amigration speed becomes slowest), and the SS mobility-conversing dot (adot where the migration speed is conversed to that of a single-strandedDNA) as featuring points. These featuring points cannot be observedunder a given condition for electrophoresis. It is preferable thatstandard DNA allows clearly observing featuring points such as themelting-starting point, the slowest point, and the SSmobility-conversing point under an adopted electrophoresis condition forgiving a standard point. In addition, it is preferable that the standardDNA gives a pattern which can be easily distinguished from that ofdouble-stranded DNA which is derived from the genome which is to beidentified.

Standard DNA should be a set of DNA (double-stranded DNA, hair pin-typeDNA/RNA, or the like) which gives clear transition near each of bothends, for example, within a predetermined temperature gradient (orconcentration gradient), and be one which is not overlapped in thetransition range of 200-1,000 base long for normally observing thetransition. Two kinds or more of standard DNA can be used for providingmore accurate standard point(s).

Not only a sequence shown by SEQ ID Nos. 1 or 2 in Sequence Listing butalso other sequences can be used for standard DNA.

Electrophoresis patterns which were obtained by TGGE with standard DNAsof SEQ ID Nos. 1 and 2 are illustrated in FIG. 1, and their propertiesare summarized in Table 1. The condition for the above-mentioned TGGEwas as follows: an electrophoresis apparatus (Taitec TG-180,manufactured by Taitec, Japan), 4% polyacrylamide gel (8M urea, 40 mMTris buffer (pH8.0)), linear temperature gradient from 30° C. to 70° C.,300V, and 90 min.

TABLE 1 SEQ No. 1 SEQ No. 2 Restored T_(i) (° C.) 60 70 temperatureT_(p) (° C.) 67 72(3) Extraction of Species Identification Dot from ElectrophoresisPattern

Bands (electrophoresis pattern) of DNA (including standard DNA) on anelectrophoresis gel which were obtained by TGGE or DGGE are visualized,for example, by silver staining, and identification dots are extractedfrom each visualized pattern.

Silver staining of the gel can be carried out, for example, according toBoulikas and Hancock (T. Boulikas, R. Hancock, J. Biochem. Biophys.Methods, 5, 219 (1981)) with an improvement using PEG treatmentaccording to Ohsawa and Ebata (K. Ohsawa, N. Ebata, Anal. Biochem., 135,409 (1983)).

The method will be outlined below:

-   1) Gel which is attached to a gel bond film is transferred to a    plastic container which contains 200 ml of a 30% PEG2000 aqueous    solution, and the obtained mixture is stirred at room temperature    (15-30° C.) for 30 min. Meanwhile the gel is separated from the    film, is shrunk, and becomes white and translucent.-   2) The solution is once substituted for 150 ml of distilled water,    and the gel is rinsed adequately. This procedure is repeated twice.    The rinsed liquid is completely sucked with an aspirator.-   3) After the rinsed liquid is removed, about 200 ml of silver stain    solution (10 ml of 1M NaOH and 2 ml of 25% ammonia are added to 200    ml of bidistilled water, and mixed, and 0.4 g of silver nitrate is    added to the mixture) is poured, and the resultant mixture is    stirred.-   4) The stain solution is substituted for 200 ml of bidistilled    water, and the water is stirred for 1 min, and the water is    discarded, and this procedure is repeated again.-   5) The liquid is substituted for about 200 ml of a developer (which    was prepared by adding 200 μl of 10% citric acid and 200 μl of    formalin to 200 ml of bidistilled water, followed by mixing), and    the gel is gently shaken until the bands are stained so as to have    appropriate density.-   6) When the bands are stained so as to have appropriate density, the    developer is quickly removed, and 200 ml of a stop solution which    was previously prepared (10% acetic acid+40% methanol, in water) is    added, and the gel and the liquid are shaken for about 10 min to    give a stained gel specimen (the waste developer should not be dried    because explosive fulminating silver can be formed).

Several ng of DNA fragments (if it is 21 ng and 300 base long, it isabout 10 fmol, i.e., 6×10⁹ molecules) which exist in 100 μl of PCRsolution can be easily detected by the treatment above (even one-tenthof that can be detected under a good condition).

In case an enough amount of DNA is available (several tens ng/band),EtBr (ethidium bromide) method can be applied, i.e., a gel afterelectrophoresis is washed with water, soaked in a 5 μg/ml EtBr solution(for about 10 min), and directly observed under a UV lamp (at about 360nm).

Alternately, species identification dots can be extracted by means of afluorescent marker carried by DNA, i.e., by using a raw material labeledwith a fluorescent marker for random PCR to amplify DNA labeled with thefluorescent marker in the amplification process by random PCR of step 1.The raw material with a fluorescent marker can be a primer or nucleotidewhich becomes a substrate for DNA polymerase. Both a primer labeled witha fluorescent marker and a polynucleotide labeled with a fluorescentmarker are well-known, and are easily commercially available.

Using a raw material for random PCR, for example, a primer labeled witha fluorescent marker, subjecting to electrophoresis DNAs which wereseparately amplified using fluorescent markers being different inexcitation and fluorescence wavelengths on one plate, and using anexcitation wavelength and a fluorescence wavelength which have differentpatterns for each DNA allows separately detecting each of the DNAs.Specifically, the obtained amplified DNA can be detected using primersA, B, and C for amplifying DNAs of organisms a, b, and c, respectively,which belong to different species one another, wherein primers A, B, andC have the same sequence, but have different fluorescent markers, usingan excitation wavelength and a fluorescence wavelength corresponding tothe fluorescent marker of primer A with respect to organism a; using anexcitation wavelength and a fluorescence wavelength corresponding to thefluorescent marker of primer B with respect to organism b; and using anexcitation wavelength and a fluorescence wavelength corresponding to thefluorescent marker of primer B with respect to organism c allowdetection of each DNA. Alternately, amplification of DNAs of an organismis made by using primers A, B, and C which are different in the sequenceand fluorescent marker and the obtained amplified DNAs can be detectedusing an excitation wavelength and a fluorescence wavelengthcorresponding to the fluorescent marker of primer A; using an excitationwavelength and a fluorescence wavelength corresponding to thefluorescent marker of primer B; and using an excitation wavelength and afluorescence wavelength corresponding to the fluorescent marker ofprimer C.

Herein ‘identification dots’ refers to inflection dot, isomobility dot,and the like which each pattern (curve) has.

Identification dots (e.g., inflection dot and isomobility dot) can bechosen, for example, in the following way:

-   1) A dot where melting begins (T_(i)) is determined as the maximal    value of second derivative or the middle dot between two dots which    give second derivatives of 0.-   2) A discontinuous dot (dot which gives a large change in Y for a    slight increase in X) is determined to give T_(min) (minimal    mobility dot).-   3) Dot Tc where a DNA arrives at a mobility of a single-stranded    state (dot where a DNA first arrives at the final mobility) is    determined.

The above-mentioned operation comprising steps 1-3 is carried out from aclear band by turns to give, for examples, 10-12 dots as a wholealthough the number of band can be appropriately chosen. If the speciesis already known and the band which corresponds to the species is known,the above-mentioned operation can be carried out with the previouslyspecified band(s).

Specifically, identification dot(s) can be extracted by the imageprocessing using a CCD camera and a computer. For the image processing,an image of an electrophoresis pattern which was visualized by theabove-mentioned silver staining is imported via an appropriate means fortaking picture into an appropriate electric recording medium as a colorimage or a gray-scale image. Specifically, an image of anelectrophoresis pattern can be imported into a computer as a genomeprofiling image using a means for taking picture such as digital camera.FIG. 1 illustrates an example of genome profiling image of a sequencefor inner standard as described below.

Genome profiling images are normalized on a computer, if necessary,after distortion is corrected. Correcting distortion can be omitted ifthe distortion of a genome profiling image is within a normal errorrange. Normalization is the image processing in which ordinate (Y-axis;mobility) and abscissa (X-axis; temperature or concentration) directionsare normalized based on species identification dots of an inner standardreference specimen, experimental condition parameters, and so on. Thisnormalization makes mobility M dimensionless so as to have a value from0 to 1. For temperature T, effective temperature which is obtained byconverting a denaturation efficiency of urea which is present in a gelinto temperature, i.e., restored temperature is applied.

After normalization, lines are extracted, and the extracted lines arefunctionalized. Lines (bold lines) are extracted by so-calledream-forming reference dot shift method with respect to each of somebands in genome profiling images. In case a band of interest iscontinuous before and after denaturation, a bold line can be extractedby specifying a dot on the band. In case a band of interest isdiscontinuous before and after denaturation, however, a bold line isextracted by specifying two dots before and after denaturation. A boldline can also be extracted using the binarization method instead of theream-forming reference dot shift method.

A bold line corresponding to each band which was extracted from a genomeprofiling image is then slimmed. Slimming is a process in which a boldline is converted into a slimmed line which is a single-valued function.In slimming, a slimmed line is obtained which consists of smooth chain(curve) which consists of many centers of gravity by the smoothingprocess in which an average coordinate of Y-coordinate of an gravity andY-coordinate of two adjacent gravities is replaced for the Y-coordinateof the gravity, for example, by the running average method.

The slimmed line is functionalized. In the functionalization, eachslimmed line is approximated to an n-order function which has anappropriate order (e.g., ten-order) or trigonometric function using, forexample, the Newton-Rapson method.

Featuring points were extracted for slimed lines and for each band curvewhich has been functionalized. Extracted featuring points includemelting-initiation point P_(int), minimal mobility point P_(min), andmobility end point P_(end). Melting-initiation point is a point on aband curve which corresponds to the initiation of melting beforedenaturation. Minimal mobility point P_(min) is a point on a band curveat which the mobility reaches the minimum. Mobility end point P_(end) isa point on a band curve at which a DNA reaches the mobility in asingle-stranded state after denaturation.

FIG. 2 illustrates a mode of the first typical band curve.

As shown in FIG. 2, in typical first band curve 51, mobility M increasesnearly linearly with temperature T at lower than the temperature(T_(ini)) at which the melting begins. The temperature reaches thetemperature (T_(ini)) which corresponds to melting initiation pointP_(ini), melting began, and mobility M decreases nearly linearly withtemperature T, and the decreasing rate is slowly decreased, and a DNAreaches minimal mobility point P_(min).

After the temperature reaches the temperature (T_(min)) whichcorresponds to minimal mobility point P_(min), mobility M rapidlyincreases with the increase in the temperature, and a DNA reaches themobility in a single-stranded state after denaturation, i.e., mobilityend P_(end). At a temperature higher than the temperature (T_(end))which corresponds to mobility end point P_(end), mobility M increasesnearly linearly with temperature T again. It is considered to be due tothe decrease in the viscosity accompanied with the increase in thetemperature, but substantially not due to the change in the shape. It isfrequently observed that minimal mobility point P_(min) becomes adiscontinuous point, and a band curve becomes discontinuous between thetemperature (T_(min)) which corresponds to minimal mobility pointP_(min) and a temperature which is slightly lower than the temperature(T_(end)) which corresponds to mobility end point P_(end).

FIG. 3 illustrates a shape of the second typical band curve.

As shown in FIG. 3, in typical second band curve 61, as similarly as inthe case of first band curve 51, mobility M increases nearly linearlywith temperature T at a temperature which is lower than the temperature(T_(ini)) at which melting begins, and the temperature reaches thetemperature (T_(ini)) which corresponds to melting initiation pointP_(ini), and melting begins, and mobility M decreases nearly linearlywith temperature T. Apart from the case of first band curve 51, however,after a DNA reaches minimal mobility point P_(min), mobility M increasesnearly linearly with temperature T again at a temperature which ishigher than the temperature (T_(min)) which corresponds to minimalmobility point P_(min). Namely, in second band curve 61, minimalmobility point P_(min) and mobility end point P_(end) conform, so that aband curve does not become discontinuous.

Based on the information such as the first differentiation, the seconddifferentiation, and its discontinuous points of band curves which wereobtained via functionalization, featuring points such as meltinginitiation point P_(ini), minimal mobility point P_(min), and mobilityend P_(end) are extracted.

Specifically, melting initiation point P_(ini) is extracted as themiddle point of a curve part which is surrounded by two straight lineparts 52 (62) and 53 (63). Hence, melting initiation point P_(ini) isdetermined based on the first differentiation value and the seconddifferentiation value of band curve 51 (61).

Minimal mobility point P_(min) is extracted, for example, asdiscontinuous points on a curve in the case of first band curve 51. Onthe other hand, in the case of second band curve 61, minimal mobilitypoint P_(min) is determined as the same point as mobility end pointP_(end) as described later.

In addition, mobility end point P_(end) is determined as a dot whichdeparts from the straight line part 54 (64) as observed along -xdirection based on the first differentiation value and the seconddifferentiation value of band curve 51 (61).

The above-mentioned featuring points extraction is carried out one byone from a clear band, i.e., a clearly subdivided band curve to give apredetermined number of featuring points as a whole. In case the speciesof a specimen microorganism is already known, and specific bands whosefeaturing points should be extracted are evident, featuring pints ofthese specific bands are extracted. FIG. 4 illustrates eight featuringpoints P_(j) (j=1-8) which were extracted from three band curves.Actually, eight featuring points or more are extracted from three bandcurves or more.

Although featuring points are extracted from band curves which wereobtained via slimming and functionalization in the above-mentioneddescription, featuring points can also be extracted directly from eachbold line without slimming or functionalization.

(4) Determination of PaSS and/or Genome Semi-distance fromIdentification Dots (temperature and mobility)

‘PaSS’ stands for Pattern Similarity Score. ‘Genome semi-distance’ is anindex which expresses a similarity of genomes of two microorganismsusing PaSS, and is expressed as (1−PaSS)/PaSS. PaSS and genomesemi-distance are determined from featuring points (spiddos) which wereobtained by the above-mentioned extraction as described below. Amicroorganism can be identified by means of genotype using PaSS and/orgenome semi-distance. A microorganism is identified by comparingfeaturing points which were obtained with a genome of a microorganism ofinterest with those of reference genome.

In case the species of a microorganism of interest is already known, thespecies is used as the reference.

In case the species of a microorganism of interest is unknown,representative species (e.g., several tens species) which werepreviously chosen based on the overall shape of the genome profilingimage are used as tentative references.

In case the species of a specimen microorganism is unknown, species aresuccessively listed up which have similar featuring points with respectto each featuring point which was obtained by the above-mentionedfeaturing point extraction (e.g., the difference in distance fromstarting point O to each featuring point is 5% or less in FIG. 7), andthe species which were listed up are assumed to be tentative references.

Then, based on featuring points of reference genomes, correspondingfeaturing points (e.g., ten dots) are determined from the featuringpoints which were obtained by the above-mentioned featuring pointsextraction, wherein ‘corresponding featuring points’ means featuringpoints which locally correspond to each of featuring points of referencegenomes. Corresponding featuring points can be determined, for example,by 1) ‘the operator specification method’ in which an operator manuallyspecifies corresponding featuring points one by one on a computerdisplay, 2) ‘the automatic assignment method’ in which a coordinate zone(a region which occupies a two-dimensional specific area in XYcoordinate) is determined to which each of featuring points of referencegenomes belongs, and specimen featuring points which belong to thecoordinate zone are successively and automatically assigned ascorresponding featuring points, 3) ‘the optimally correspondingautomatic calculation method’ in which the number of correspondingfeaturing points are appropriately set, and featuring points arearbitrarily chosen from featuring points of reference genomes andextracted specimen featuring points so that the number of the featuringpoints can be each of the numbers of corresponding featuring points, andthey are associated according to the combination theory, andcorresponding featuring points are automatically determined based on thecombination which gives the largest PaSS which is to be defined later.

In this way, as shown in FIG. 5, ‘n’ featuring points P_(1i) (i=1−n) ofa specimen microorganism are obtained corresponding to ‘n’ featuringpoints P_(0i) (i=1−n) of a reference genome, wherein each featuringpoint P_(i) (‘n’ featuring points P_(0i) and ‘n’ featuring points of aspecimen microorganism) has position vector V_(i) (position vectorV_(0i) of ‘n’ featuring points of a reference genome and position vectorV_(1i) of ‘n’ featuring points of a specimen microorganism). Point O atwhich T=the lowest temperature at M=0 can be used as the starting pointof position vector V_(i).

Using position vector V_(i) of each featuring point P_(i), PaSS isobtained which is defined by Eq.1 below:PaSS=1−{Σγ(i)}/n  (1)where ‘Σ’ denotes the sum of i=1-n, and γ(i) is expressed by Eq.2 below:γ(i)=2×|V _(1i) −V _(0i)|/(|V _(1i) |+|V _(0i)|)  (2)

Although a value of PaSS is determined according to the vector(dependent) type calculation method in the above-mentioned description,the value can also be determined based on the scalar type calculationmethod as describe below:PaSS=1−{Σr(i)}/n  (4)where ‘Σ’ denotes the sum of i=1−n, and r(i) is expressed by Eq.5 below:r(i)=[[T _(1i) −T _(0i)]/(T _(w)/α)]²+[(M _(1i) −M _(0i))/(M _(1i) +M_(0i))/2]²]^(½)/(α²+1²)^(½)  (5)where ‘T_(0i)’ denotes a temperature of ‘n’ featuring points P_(0i) of areference genome, and ‘T_(1i)’ denotes a temperature of ‘n’ featuringpoints P_(1i) of a specimen organism. ‘M_(0i)’ denotes the mobility of‘n’ featuring points P_(0i) of a reference genome, and ‘M_(1i)’ denotesthe mobility of ‘n’ featuring points P_(1i) of a specimen organism.‘T_(w)’ denotes the temperature normalization factor which correspondsto a temperature range (e.g., about 60° C.) which gives featuringpoints. ‘α’ denotes a relative value of both ‘weights’ of mobilitychange and temperature change, and is normally 1. Denominator part(α²+1²)^(½) in Eq.5 constitutes the second normalization factor (so thatan r(i) value can be 1 or less).

Using PaSS which is obtained based on Eq.1 or 4, genome semi-distanced_(s) is obtained which is defined by Eq.3 below:d _(s)=(1−PaSS)/PaSS  (3)

In general, in case the species of a specimen microorganism is the sameas that of a reference genome, a PaSS value (score) becomes nearly 1,and a genome semi-distance d_(s) value becomes nearly 0.

(5) Identification of Microorganism Based on PaSS and/or GenomeSemi-distance

In case a species of a microorganism of interest is unknown, PaSS, andif necessary, genome semi-distance d_(s) is/are calculated for eachreference genome. Then, comparison with other reference genome isrepeated until a PaSS value goes over a standard value (e.g., 0.96) or agenome semidistance d_(s) value goes below a standard value (e.g.,0.04). In this way, a species of a specimen microorganism can beidentified based on a PaSS value or a genome semi-distance d_(s) value.

On the other hand, in case the species of a microorganism which is to beidentified is known, similarity between individuals can be determinedwhich belong to the same species based on a PaSS or genome semi-distanced_(s) value. Namely, PaSS or genome semi-distance d_(s) is determinedfor two specimen microorganisms which belong to the same species, and incase two PaSS values are adequately near or two genome semi-distanced_(s) values are adequately near, it is determined that the two specimenmicroorganisms belong to the same species and similarity between theindividuals is high.

In addition, it is preferable to store by plotting a set of species orindividuals which exist within a specific genome semi-distance in thegenome sequence space as neighboring information.

In this way, it is preferable to resister genome profiling images,featuring points, judgment information, neighboring information, and soon as an appropriate data base, and constitute them so as to beavailable at any time.

As described above, a melting initiation point which corresponds to theinitiation of melting before denaturation, a minimal mobility pointwhere the mobility reaches the minimum before strand dissociation, and amobility end point where a DNA first reaches the mobility in asingle-stranded state after denaturation are extracted as featuringpoints from a genome profiling imaged. As a result, by a quantitativemethod using the obtained featuring points, a species of a microorganismand similarity between individuals which are classified into the samespecies of microorganism can be accurately and simply identified.

A method for identifying a species of and a method for identifyingsimilarity of a microorganism by its genotype are described above. Amethod according to the present invention is not limited to amicroorganism, but is also applicable to methods for identification suchas a method for identifying a species of and a method for identifyingsimilarity of a general organism.

EXAMPLES

The present invention will be described in more detail by examplesbelow.

Example 1

Genome profiling (GP) according to the present invention of strains ofyeasts, Escherichia coli, and Bacillus subtilis, and mutants therefrom(twenty kinds as a whole) was carried out as described below, and theirspiddos were determined with a computer, and were registered as adatabase. Bacillus subtilis was newly isolated from commerciallyavailable natto, and DNA was extracted, and GP was carried out to givespiddos, which were assumed as spiddos of a tentative unknown specimenX.

Preparation of Double-stranded DNA Fragment by Random PCR

One hundred μl of reaction mixture which contains 200 μM dNTP (N=G, A,T, C), 0.5 μM primer(pfM12 (dAGAACGCGCCTG) (SEQ ID NO: 39)) 10 mMTris-HCl (pH 9.0), 50 mM KCl, 2.5 mM MgCl₂, 0.1% Triton X-100, 0.02unit/μl Taq DNA polymerase, and an appropriate amount of template DNA (3μl of a DNA solution was added, and the volume of the reaction mixturewas adjusted to 100 μl ) was prepared for each DNA. Double-stranded DNAfragments were prepared by PCR comprising the steps of 1) the treatmentat 94° C. for 1 min, 2) 20-30 times repeating the cycle comprisingdenaturation at 94° C. for 30 min, annealing at 28° C. for 2 min, andelongation at 47° C. for 2 min, followed by 3) the treatment at 47° C.for 2 min.

TGGE of Double-stranded DNA Fragment

To the double-stranded DNA fragment which was obtained as describedabove, 0.3 μg of DNA which has SEQ ID No.1 was added as standard DNA,and TGGE was carried out. Condition for TGGE is as follows: 4%polyacrylamide gel (40 mM Tris buffer (pH 8.0) which contains 8M urea),linear temperature gradient from 30 to 70° C., 400V, 110 min, Taitecelectrophoresis apparatus TG-180.

Extraction of Identification Dots of Each DNA Fragment fromElectrophoresis Pattern

A band (electrophoresis pattern) of DNA (including standard DNA) on anelectrophoresis gel which was obtained by TGGE was silver stained asdescribed below, and identification dots were extracted from eachvisualized pattern.

-   1) A gel which is attached to a gel bond film is directly    transferred into a plastic container which contains 200 ml of a 30%    PEG 2000 aqueous solution, and stirred at room temperature (15-30°    C.) for 30 min. Meanwhile, the gel is detached from the film,    contracted, and becomes white and translucent.-   2) The solution is once substituted for 150 ml of distilled water,    and the gel is adequately rinsed. This procedure is repeated again.    Rinsed water is absolutely sucked with an aspirator.-   3) After the rinsed water is removed, about 200 ml of a silver stain    solution (which was prepared by adding 10 ml of 1M NaOH and 2 ml of    25% ammonia aqueous solution to 200 ml of bidistilled water, mixing,    followed by dissolving 0.4 g of silver nitrate) is added, and    stirred for 30 min.-   4) The liquid is substituted for 200 ml of bidistilled water, and    stirred for 1 min, and the water is discarded. This procedure is    repeated again.-   5) The liquid is substituted for about 200 ml of a developer (which    was prepared by adding 200 μl of 10% citric acid and 200 μl of    formalin to 200 ml of bidistilled water, followed by dissolving),    and gently shaken until a band is appropriately stained.-   6) When the band is appropriately stained, the developer is quickly    removed, and200 ml of a previously prepared stop solution (10%    acetic acid+40% methanol, in water) is added, and stirred for about    10 min to give a stained gel specimen.

Featuring points were extracted from each pattern which was visualizedby the above-mentioned method, and spiddos were determined.Identification dots were extracted as follows:

A gel picture of GP was imported into a computer with a scanner, and theobtained image was corrected and normalized with a computer, andidentification dots were extracted by a method how featuring points werespecified for an image on a display with a mouse.

The spiddos which were obtained in this way of an unknown specimen werecompared with ten spiddos of various genomes on a data base, and PaSSwas calculated by the total combination calculation method.

A genome which gives the highest PaSS with X was automatically extractedfrom 20 kinds of genomes which were previously registered.

Results were obtained as a computer output, and accompanying informationrevealed that the organism which X showed the highest similarity isBacillus subtilis.

Example 2

Random PCR was carried out under the same condition except using DNA ofBacillus subtilis as a template, and cy3-pfM12 (5′ cy3-AGA ACG CGC CTG3′) (SEQ ID NO: 4) which contains phosphor cy3 or FITC/UCS (5′FITC-CAGGAAACGGCTATGGAC3′) (SEQ ID NO: 40) which contains phosphor FITCas a primer for each primer, and the obtained double-stranded amplifiedDNA fragments were mixed, and TGGE was carried out in a way which issimilar to Example 1. An electrophoresis pattern by TGGE was visualizedwith an excitation/fluorescent wavelength of 550 nm/570 nm (phosphorcy3) or 494 nm/519 nm (phosphor FITC). FIG. 6 a illustrates anelectrophoresis pattern in the case where cy3-pfM12(5′cy3-AGAACGCGCCTG3′) (SEQ ID NO: 4) was used as a primer, and FIG. 6 billustrates an electrophoresis pattern in the case where FITC/UCS (5′FITC-CAGGAAACAGCTATGAC3′) was used as a primer. Identification dots wereextracted form each pattern. The microorganism which was used as atemplate was identified as Bacillus subtilis based the obtainedidentification dots.

The present disclosure relates to the subject matter contained inJapanese Patent Application No. 2000-123755, filed on Apr. 25, 2000,which is expressly incorporated herein by reference in its entirely.

While illustrative and presently preferred embodiments of the presentinvention have been described in detail herein, it is to be understoodthat the inventive concepts may be otherwise variously embodied andemployed and that the appended claims are intended to be construed to beconstrued to include such variations except insofar as limited by theprior art.

1. A method for determining the similarity at the genome level between atarget organism and a reference organism comprising the steps of: a)preparing one or more of double-stranded DNA fragments by random PCRusing, as a template, genome DNA of the target organism, b) subjectingsaid double-stranded DNA fragments prepared in step a) to temperaturegradient gel electrophoresis (TGGE) or denaturant gradient gelelectrophoresis (DGGE), wherein an internal reference double-strandedDNA prepared in advance, is co-migrated with the double-stranded DNAfragments, c) assigning each melting initiation point and/or eachmobility transition end point of the double-stranded DNA fragmentsprepared in step a) and both the melting initiation point and themobility transition end point of the internal reference double-strandedDNA co-migrated from an electrophoretic pattern obtained in step b), d)normalizing each coordinate of melting initiation point and/or mobilitytransition end point of the double-stranded DNA fragments using those ofthe melting initiation point and mobility transition end point of theinternal reference double-stranded DNA to eliminate experimentalfluctuations and generate species identification dots, e) calculatingPaSS (Pattern Similarity Score) defined by equation 1 below:PaSS=1−{Σγ(i)}/n  (1) where ‘Σ’ denotes the summation over i=1 to n(where n is the number of the species identification dots used for thecalculation), and γ(i) is expressed by equation 2 below:γ(i)=2×|V _(1i) −V _(0i)|/(|V _(1i) |+|V _(0i)|)  (2) where V_(0i)represents position vector of the i-th species identification dots ofthe reference organism and V_(1i) represents position vector of the i-thspecies identification dots of the target organism; using speciesidentification dots obtained in step d) and species identification dotsof the reference organism to determine the similarity at the genomelevel between the target organism and the reference organism, whereinthe species identification dots of the reference organism is separatelyobtained by a method in which steps a) to d) are carried out under thesame conditions.
 2. A method according to claim 1, wherein said standardDNA is SEQ ID NO: 1 or SEQ ID NO:
 2. 3. A method according to claim 1,wherein said identification of an organism is the species identificationor homology identification of an organism.
 4. A method according toclaim 1, wherein in step a), a primer or nucleotide labeled with afluorescent marker is used for said random PCR to amplify DNA fragmentswith a fluorescent marker and a fluorescence labeled DNA is used as thestandard DNA, and in step c), said extraction from the featuring pointsis carried out by image processing using the fluorescent markers carriedby the DNAs.
 5. A method according to claim 1, wherein the featuringpoints which are obtained in step c) are expressed by the coordinates ofthe temperature axis and the mobility axis in the case of temperaturegradient gel electrophoresis (TGGE), and by the coordinates of thedenaturant concentration axis and the mobility axis in the case ofdenaturant gradient gel electrophoresis (DGGE).
 6. A method according toclaim 1, wherein said organism is a microorganism.
 7. A method foridentifying a reference organism closest at the genome level to a targetorganism comprising the steps of: a) preparing one or more ofdouble-stranded DNA fragments by random PCR using, as a template, genomeDNA of the target organism, b) subjecting said double-stranded DNAfragments prepared in step a) to temperature gradient gelelectrophoresis (TGGE) or denaturant gradient gel electrophoresis(DGGE), wherein an internal reference double-stranded DNA prepared inadvance, is co-migrated with the double-stranded DNA fragments, c)assigning each melting initiation point and/or each mobility transitionend points of the double-stranded DNA fragments prepared in step a) andboth the melting initiation point and the mobility transition end pointof the internal reference double-stranded DNA co-migrated from anelectrophoretic pattern obtained in step b), d) normalizing eachcoordinate of melting initiation point and/or mobility transition endpoint of the double-stranded DNA fragments using those of the meltinginitiation point and/or mobility transition end point of the internalreference double-stranded DNA to eliminate experimental fluctuations andgenerate species identification dots, e) calculating PaSS (PatternSimilarity Score) defined by equation 1 below:PaSS=1−{Σγ(i)}/n  (1) where ‘Σ’ denotes the summation over i=1 to n(where n is the number of the species identification dots used for thecalculation), and γ(i) is expressed by equation 2 below:γ(i)=2×|V _(1i) −V _(0i)|/(|V _(1i) |+|V _(0i)|)  (2) where V₀₁represents position vector of the i-th species identification dots ofthe reference organism and V_(1i) represents position vector of the i-thspecies identification dots of the target organism; using speciesidentification dots obtained in step d) and species identification dotsof a reference organism deposited in a database in which speciesidentification dots of organisms separately determined by a method inwhich steps a) to d) are carried out under the same conditions areregistered, and f) repeating calculation of PaSS of step e) withaltering the reference organism until the maximum PaSS is reached toidentify the reference organism closest at the genome level to thetarget organism.
 8. A method according to claim 7, wherein said standardDNA is SEQ ID NO: 1 or SEQ ID NO:
 2. 9. A method according to claim 7,wherein said identification of an organism is the species identificationor homology identification of an organism.
 10. A method according toclaim 7, wherein in step a), a primer or nucleotide labeled with afluorescent marker is used for said random PCR to amplify DNA fragmentswith a fluorescent marker and a fluorescence labeled DNA is used as thestandard DNA, and in step c), said extraction from the featuring pointsis carried out by image processing using the fluorescent markers carriedby the DNAs.
 11. A method according to claim 7, wherein the featuringpoints which are obtained in step c) are expressed by the coordinates ofthe temperature axis and the mobility axis in the case of temperaturegradient gel electrophoresis (TGGE), and by the coordinates of thedenaturant concentration axis and the mobility axis in the case ofdenaturant gradient gel electrophoresis (DGGE).
 12. A method accordingto claim 7, wherein said organism is a microorganism.
 13. A method fordetermining the similarity at the genome level between a target organismand a reference organism comprising the steps of: a) preparing one ormore of double-stranded DNA fragments by random PCR using, as atemplate, genome DNA of the target organism, b) subjecting saiddouble-stranded DNA fragments prepared in step a) to temperaturegradient gel electrophoresis (TGGE) or denaturant gradient gelelectrophoresis (DGGE), wherein an internal reference double-strandedDNA prepared in advance, is co-migrated with the double-stranded DNAfragments, c) assigning each melting initiation point and/or eachmobility transition end point of the double-stranded DNA fragmentsprepared in step a) and both the melting initiation point and themobility transition end point of the internal reference double-strandedDNA co-migrated from an electrophoretic pattern obtained in step b), d)normalizing each coordinate of melting initiation point and/or mobilitytransition end point of the double-stranded DNA fragments using those ofthe melting initiation point and mobility transition end point of theinternal reference double-stranded DNA to eliminate experimentalfluctuations and generate species identification dots, e) calculatinggenome semi-distance by the formula(1−Pass)/Pass to determine the similarity at the genome level betweenthe target organism and the reference organism, PaSS (Pattern SimilarityScore) defined by equation 1 below:PaSS=1−{Σγ(i)}/n  (1) where ‘Σ’ denotes the summation over i=1 to n(where n is the number of the species identification dots used for thecalculation), and γ(i) is expressed by equation 2 below:γ(i)=2×|V _(1i) −V _(0i)|/(|V _(1i) |+|V _(0i)|)  (2) where V_(0i)represents position vector of the i-th species identification dots ofthe reference organism and V_(1i) represents position vector of the i-thspecies identification dots of the target organism; using speciesidentification dots obtained in step d) and species identification dotsof the reference organism to determine the similarity at the genomelevel between the target organism and the reference organism, whereinthe species identification dots of the reference organism is separatelyobtained by a method in which steps a) to d) are carried out under thesame conditions.
 14. A method according to claim 13, wherein saidstandard DNA is SEQ ID NO: 1 or SEQ ID NO:
 2. 15. A method according toclaim 13, wherein said identification of an organism is the speciesidentification or homology identification of an organism.
 16. A methodaccording to claim 13, wherein in step a), a primer or nucleotidelabeled with a fluorescent marker is used for said random PCR to amplifyDNA fragments with a fluorescent marker and a fluorescence labeled DNAis used as the standard DNA, and in step c), said extraction from thefeaturing points is carried out by image processing using thefluorescent markers carried by the DNAs.
 17. A method according to claim13, wherein the featuring points which are obtained in step c) areexpressed by the coordinates of the temperature axis and the mobilityaxis in the case of temperature gradient gel electrophoresis (TGGE), andby the coordinates of the denaturant concentration axis and the mobilityaxis in the case of denaturant gradient gel electrophoresis (DGGE). 18.A method according to claim 13, wherein said organism is amicroorganism.
 19. A method for identifying a reference organism closestat the genome level to a target organism comprising the steps of: a)preparing one or more of double-stranded DNA fragments by random PCRusing, as a template, genome DNA of the target organism, b) subjectingsaid double-stranded DNA fragments prepared in step a) to temperaturegradient gel electrophoresis (TGGE) or denaturant gradient gelelectrophoresis (DGGE), wherein an internal reference double-strandedDNA prepared in advance, is co-migrated with the double-stranded DNAfragments, c) assigning each melting initiation point and/or eachmobility transition end points of the double-stranded DNA fragmentsprepared in step a) and both the melting initiation point and themobility transition end point of the internal reference double-strandedDNA co-migrated from an electrophoretic pattern obtained in step b), d)normalizing each coordinate of melting initiation point and/or mobilitytransition end point of the double-stranded DNA fragments using those ofthe melting initiation point and/or mobility transition end point of theinternal reference double-stranded DNA to eliminate experimentalfluctuations and generate species identification dots, e) calculatinggenome semi-distance by the formula(1−Pass)/Pass to determine the similarity at the genome level betweenthe target organism and the reference organism, PaSS (Pattern SimilarityScore) defined by equation 1 below:PaSS=1−{Σγ(i)}/n  (1) where ‘Σ’ denotes the summation over i=1 to n(where n is the number of the species identification dots used for thecalculation), and γ(i) is expressed by equation 2 below:γ(i)=2×|V _(1i) −V _(0i)|/(|V _(1i) |+|V _(0i)|)  (2) where V_(0i)represents position vector of the i-th species identification dots ofthe reference organism and V_(1i) represents position vector of the i-thspecies identification dots of the target organism; using speciesidentification dots obtained in step d) and species identification dotsof a reference organism deposited in a database in which speciesidentification dots of organisms separately determined by a method inwhich steps a) to d) are carried out under the same conditions areregistered, and f) repeating calculation of genome semi-distance of stepe) with altering the reference organism until the maximum genomesemi-distance is reached to identify the reference organism closest atthe genome level to the target organism.
 20. A method according to claim19, wherein said standard DNA is SEQ ID NO: 1 or SEQ ID NO:
 2. 21. Amethod according to claim 19, wherein said identification of an organismis the species identification or homology identification of an organism.22. A method according to claim 19, wherein in step a), a primer ornucleotide labeled with a fluorescent marker is used for said random PCRto amplify DNA fragments with a fluorescent marker and a fluorescencelabeled DNA is used as the standard DNA, and in step c), said extractionfrom the featuring points is carried out by image processing using thefluorescent markers carried by the DNAs.
 23. A method according to claim19, wherein the featuring points which are obtained in step c) areexpressed by the coordinates of the temperature axis and the mobilityaxis in the case of temperature gradient gel electrophoresis (TGGE), andby the coordinates of the denaturant concentration axis and the mobilityaxis in the case of denaturant gradient gel electrophoresis (DGGE). 24.A method according to claim 19, wherein said organism is amicroorganism.